The long-term goal of this proposal is to establish conjugal DNA transfer in Mycobacterium tuberculosis in order to manipulate the genome of this major human pathogen. Although both transformation and transduction have had important roles in the development of mycobacterial genetics, conjugal DNA transfer has yet to be established in M. tuberculosis. Conjugation offers an important alternative for transferring DNA between mycobacteria and, in particular, as a gene delivery system for moving markers between strains and generating targeted mutations. This laboratory has established an efficient conjugal DNA transfer system in the model mycobacterial organism Mycobacterium smegmatis. Here, we propose to apply our expertise and technology to facilitate genetic manipulation of the M. tuberculosis genome. Transfer will be established from from a hyper- conjugative strain of M. smegmatis into M. tuberculosis and between strains of M. tuberculosis. The specific aims are: 1. To establish chromosomal DNA transfer into M. tuberculosis from M. smegmatis utilizing a combination of genetic selections and counter-selections. 2. To optimize plasmid transfer into M. tuberculosis in order to develop a novel and efficient method for in vivo genetic engineering. 3. To create an M. tuberculosis donor that will allow transfer between strains of M. tuberculosis. The establishment of DNA transfer into M. tuberculosis would represent a significant step forward in the ability to genetically analyse this organism. Moreover, demonstration of genetic exchange would implicate conjugation as a significant player in the evolution of M. tuberculosis, and would raise the sinister possibility of drug resistance genes being exchanged between M. tuberculosis cells in the lung, thereby creating multi-drug resistant strains. Mycobacterium tuberculosis accounts for over 2 million deaths per year. Furthermore, the global burden of tuberculosis has been compounded by its deadly association with the AIDS virus and by the emergence of multi-drug resistant strains, which have increased the demand for new treatments. The design of new drugs and vaccines requires an understanding of the biology of mycobacteria, and the development of genetic tools to manipulate their genomes in order to determine the molecular basis of pathogenesis and drug resistance. [unreadable] [unreadable] [unreadable]